Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Active turnover modulates mature microRNA activity in Caenorhabditis elegans

Abstract

MicroRNAs (miRNAs) constitute a large class of regulatory RNAs that repress target messenger RNAs to control various biological processes1. Accordingly, miRNA biogenesis is highly regulated, controlled at both transcriptional and post-transcriptional levels2, and overexpression and underexpression of miRNAs are linked to various human diseases, particularly cancers1,3. As RNA concentrations are generally a function of biogenesis and turnover, active miRNA degradation might also modulate miRNA accumulation, and the plant 3′→5′ exonuclease SDN1 has been implicated in miRNA turnover4. Here we report that degradation of mature miRNAs in the nematode Caenorhabditis elegans, mediated by the 5′→3′ exoribonuclease XRN-2, affects functional miRNA homeostasis in vivo. We recapitulate XRN-2-dependent miRNA turnover in larval lysates, where processing of precursor-miRNA (pre-miRNA) by Dicer, unannealing of the miRNA duplex and loading of the mature miRNA into the Argonaute protein of the miRNA-induced silencing complex (miRISC) are coupled processes that precede degradation of the mature miRNA. Although Argonaute:miRNA complexes are highly resistant to salt, larval lysate promotes efficient release of the miRNA, exposing it to degradation by XRN-2. Release and degradation can both be blocked by the addition of miRNA target RNA. Our results therefore suggest the presence of an additional layer of regulation of animal miRNA activity that might be important for rapid changes of miRNA expression profiles during developmental transitions and for the maintenance of steady-state concentrations of miRNAs. This pathway might represent a potential target for therapeutic intervention on miRNA expression.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Depletion of xrn-2 increases mature miRNA levels and activity.
Figure 2: Coordination of in vitro miRNA processing and turnover.
Figure 3: Target-mediated stabilization of mature miRNA.
Figure 4: Release of miRNA from miRISC.

Similar content being viewed by others

References

  1. Chang, T. C. & Mendell, J. T. microRNAs in vertebrate physiology and human disease. Annu. Rev. Genomics Hum. Genet. 8, 215–239 (2007)

    Article  CAS  Google Scholar 

  2. Ding, X. C., Weiler, J. & Großhans, H. Regulating the regulators: mechanisms controlling the maturation of microRNAs. Trends Biotechnol. 27, 27–36 (2009)

    Article  CAS  Google Scholar 

  3. Esquela-Kerscher, A. & Slack, F. J. Oncomirs—microRNAs with a role in cancer. Nature Rev. Cancer 6, 259–269 (2006)

    Article  CAS  Google Scholar 

  4. Ramachandran, V. & Chen, X. Degradation of microRNAs by a family of exoribonucleases in Arabidopsis . Science 321, 1490–1492 (2008)

    Article  ADS  CAS  Google Scholar 

  5. Büssing, I., Slack, F. J. & Großhans, H. let-7 microRNAs in development, stem cells and cancer. Trends Mol. Med. 14, 400–409 (2008)

    Article  Google Scholar 

  6. Reinhart, B. J. et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans . Nature 403, 901–906 (2000)

    Article  ADS  CAS  Google Scholar 

  7. Vella, M. C., Choi, E. Y., Lin, S. Y., Reinert, K. & Slack, F. J. The C. elegans microRNA let-7 binds to imperfect let-7 complementary sites from the lin-41 3′UTR. Genes Dev. 18, 132–137 (2004)

    Article  CAS  Google Scholar 

  8. Bagga, S. et al. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 122, 553–563 (2005)

    Article  CAS  Google Scholar 

  9. Abbott, A. L. et al. The let-7 microRNA family members mir-48, mir-84, and mir-241 function together to regulate developmental timing in Caenorhabditis elegans . Dev. Cell 9, 403–414 (2005)

    Article  CAS  Google Scholar 

  10. Kennedy, S., Wang, D. & Ruvkun, G. A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans . Nature 427, 645–649 (2004)

    Article  ADS  CAS  Google Scholar 

  11. Chernyakov, I., Whipple, J. M., Kotelawala, L., Grayhack, E. J. & Phizicky, E. M. Degradation of several hypomodified mature tRNA species in Saccharomyces cerevisiae is mediated by Met22 and the 5′–3′ exonucleases Rat1 and Xrn1. Genes Dev. 22, 1369–1380 (2008)

    Article  CAS  Google Scholar 

  12. Weidhaas, J. B. et al. MicroRNAs as potential agents to alter resistance to cytotoxic anticancer therapy. Cancer Res. 67, 11111–11116 (2007)

    Article  CAS  Google Scholar 

  13. Gy, I. et al. Arabidopsis FIERY1, XRN2, and XRN3 are endogenous RNA silencing suppressors. Plant Cell 19, 3451–3461 (2007)

    Article  CAS  Google Scholar 

  14. Lee, R. C. & Ambros, V. An extensive class of small RNAs in Caenorhabditis elegans . Science 294, 862–864 (2001)

    Article  ADS  CAS  Google Scholar 

  15. Ketting, R. F. et al. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans . Genes Dev. 15, 2654–2659 (2001)

    Article  CAS  Google Scholar 

  16. Grishok, A. et al. Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell 106, 23–34 (2001)

    Article  CAS  Google Scholar 

  17. Großhans, H., Johnson, T., Reinert, K. L., Gerstein, M. & Slack, F. J. The temporal patterning microRNA let-7 regulates several transcription factors at the larval to adult transition in C. elegans . Dev. Cell 8, 321–330 (2005)

    Article  Google Scholar 

  18. Slack, F. J. et al. The lin-41 RBCC gene acts in the C. elegans heterochronic pathway between the let-7 regulatory RNA and the LIN-29 transcription factor. Mol. Cell 5, 659–669 (2000)

    Article  CAS  Google Scholar 

  19. Ding, X. C. & Großhans, H. Repression of C. elegans microRNA targets at the initiation level of translation requires GW182 proteins. EMBO J. 28, 213–222 (2009)

    Article  CAS  Google Scholar 

  20. Stevens, A. & Poole, T. L. 5′-exonuclease-2 of Saccharomyces cerevisiae. Purification and features of ribonuclease activity with comparison to 5′-exonuclease-1. J. Biol. Chem. 270, 16063–16069 (1995)

    Article  CAS  Google Scholar 

  21. Stevens, A. & Maupin, M. K. A 5′–3′ exoribonuclease of human placental nuclei: purification and substrate specificity. Nucleic Acids Res. 15, 695–708 (1987)

    Article  CAS  Google Scholar 

  22. Hutvagner, G. et al. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science 293, 834–838 (2001)

    Article  CAS  Google Scholar 

  23. Pillai, R. S. et al. Inhibition of translational initiation by let-7 microRNA in human cells. Science 309, 1573–1576 (2005)

    Article  ADS  CAS  Google Scholar 

  24. Hutvagner, G., Simard, M. J., Mello, C. C. & Zamore, P. D. Sequence-specific inhibition of small RNA function. PLoS Biol. 2, E98 (2004)

    Article  Google Scholar 

  25. Wang, Y., Sheng, G., Juranek, S., Tuschl, T. & Patel, D. J. Structure of the guide-strand-containing argonaute silencing complex. Nature 456, 209–213 (2008)

    Article  ADS  CAS  Google Scholar 

  26. Martinez, J. & Tuschl, T. RISC is a 5′ phosphomonoester-producing RNA endonuclease. Genes Dev. 18, 975–980 (2004)

    Article  CAS  Google Scholar 

  27. Bhattacharyya, S. N., Habermacher, R., Martine, U., Closs, E. I. & Filipowicz, W. Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell 125, 1111–1124 (2006)

    Article  CAS  Google Scholar 

  28. Kedde, M. et al. RNA-binding protein Dnd1 inhibits microRNA access to target mRNA. Cell 131, 1273–1286 (2007)

    Article  CAS  Google Scholar 

  29. Ding, X. C., Slack, F. J. & Großhans, H. The let-7 microRNA interfaces extensively with the translation machinery to regulate cell differentiation. Cell Cycle 7, 3083–3090 (2008)

    Article  CAS  Google Scholar 

  30. Pall, G. S. & Hamilton, A. J. Improved northern blot method for enhanced detection of small RNA. Nature Protocols 3, 1077–1084 (2008)

    Article  CAS  Google Scholar 

  31. Hager, D. A. & Burgess, R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal. Biochem. 109, 76–86 (1980)

    Article  CAS  Google Scholar 

  32. Chatterjee, S. et al. An RNA-binding respiratory component mediates import of type II tRNAs into Leishmania mitochondria. J. Biol. Chem. 281, 25270–25277 (2006)

    Article  CAS  Google Scholar 

  33. Mathy, N. et al. 5′-to-3′ exoribonuclease activity in bacteria: role of RNase J1 in rRNA maturation and 5′ stability of mRNA. Cell 129, 681–692 (2007)

    Article  CAS  Google Scholar 

  34. Kolb, F. A. et al. Human dicer: purification, properties, and interaction with PAZ PIWI domain proteins. Methods Enzymol. 392, 316–336 (2005)

    Article  CAS  Google Scholar 

  35. Matranga, C., Tomari, Y., Shin, C., Bartel, D. P. & Zamore, P. D. Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123, 607–620 (2005)

    Article  CAS  Google Scholar 

  36. Dziembowski, A., Lorentzen, E., Conti, E. & Séraphin, B. A single subunit, Dis3, is essentially responsible for yeast exosome core activity. Nature Struct. Mol. Biol. 14, 15–22 (2007)

    Article  CAS  Google Scholar 

  37. Lee, M. H. & Schedl, T. Identification of in vivo mRNA targets of GLD-1, a maxi-KH motif containing protein required for C. elegans germ cell development. Genes Dev. 15, 2408–2420 (2001)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Bühler and W. Filipowicz for critical comments on the manuscript; W. Filipowicz for plasmids; C. Mello and F. Slack for C. elegans strains; and A. Esquela-Kerscher for sharing Starfire probes for pre-miRNA detection. S.C. was supported by Marie Curie and EMBO long-term postdoctoral fellowships.

Author Contributions S.C. and H.G. designed the research. S.C. designed and performed the experiments. S.C. and H.G. analysed the experimental results and wrote the manuscript.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Helge Großhans.

Supplementary information

Supplementary information

This file contains Supplementary Notes, Supplementary Table S1 and Supplementary Figures S1-S18 with Legends. (PDF 4930 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chatterjee, S., Großhans, H. Active turnover modulates mature microRNA activity in Caenorhabditis elegans. Nature 461, 546–549 (2009). https://doi.org/10.1038/nature08349

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08349

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing